Site Loader
Rock Street, San Francisco

1. The study broadly hypothesized that a
recombinant form of human vimentin (rhVim) acts to inhibit the leukocyte-platelet
and leukocyte-endothelium interactions via the interruption of platelet
selectins and leukocyte ligands.

2. The study looked into vimentin’s role in
blocking the above interactions because the molecule is associated with inflammation.

We Will Write a Custom Essay Specifically
For You For Only $13.90/page!

order now

Specifically, on leukocytes, vimentin was shown to bind a portion of PSGL-1,
and on neutrophils, vimentin may also bind a soluble E-selectin ligand. Interactions
between E-selectin, P-selectin (whose ligand is PSGL-1), and L-selectin have
been shown, tying vimentin interactions to the roles of neutrophils and
platelets during inflammation. Thus, vimentin is a possible inhibitor of the
inflammatory response via its disruption of neutrophil-platelet and
neutrophil-endothelium interactions, as stated in the hypothesis.

3. A recombinant form of vimentin was created
via transforming an IPTG-inducible plasmid into E.coli to use for the duration of the study. The protein was purified via elution and dialysis. Purity was
confirmed via visualization on SDS-PAGE. Western blot analysis confirmed

Blood was
collected and fluorochrome-tagged leukocytes and platelets were isolated via
flow cytometry to confirm the portion of the hypothesis that supposes soluble
vimentin has an effect on platelet-leukocyte interactions (see following

In vitro parallel-plate flow-adhesion was used to
image the capture of neutrophils/leukocytes labelled with mepacrine as they
flowed across fibrin(ogen), platelet, or human umbilical vein endothelial cells
(HUVECs) monolayers. Capture was measured. This was used to confirm the hypothesis
supposition that rhVim inhibits leukocyte-platelet and leukocyte-endothelium
interactions by measuring the interactions in the presence of rhVim or not

HUVECs were
stimulated to express P-selectin, confirming the hypothesis supposition that
platelet selectin interactions, notably PSGL-1-P-selectin, may be the target of

The binding
assay consisted of immunoplates against either P-selectin or PSGL-1. RhVim was
added to the plate, incubated, and washed. Leftover rhVim was detected with a horseradish
peroxidase-conjugated antibody and tetramethylbenzidine substrate to
confirm the hypothesis supposition that rhVim disrupts leukocyte-platelet
interactions. It also finds rhVim is specific against P-selectin, proving the
hypothesis that rhVim disrupts selectin-ligand interaction.

In vitro parallel-plate flow-adhesion was again
performed to find rhVim dose-dependently binds to P-selectin as opposed to
PSGL-1. This again confirms leukocyte-platelet interruption.

plasmon resonance used optics to look at the binding of various mobile proteins
over an immobile rhVim to measure KD, a measure of binding affinity,
of P-selectin and E-selectin to rhVim. This again proved the rhVim interrupts
selectin-ligand interactions. Bio-layer interferometry looks at binding
kinetics of the above interactions and proved the same point.

endotoxin-induced murine acute lung injury (ALI) model included the injection
of mice with rhVim then LPS. Mice were evaluated for sepsis and their lungs
fixed and analyzed for signs of lung injury including infiltration of
neutrophils, alveolar wall thickness, and alveoli filling of fluid along with
other indications of inflammation to determine if rhVim does decrease leukocyte
infiltration into the lungs. This proves rhVim treatment decreases signs of inflammation
in vivo.

4. This paper builds from the lecture topic
that inflammation, in part attributed to leukocyte migration, may cause disease;
thus, inflammation attenuation may be a key therapeutic method. The 3 papers advertise
attenuation of leukocyte migration inhibition via leukocyte-platelet
communication (notably PSGL-1-P-selectin) inhibition as a solution to
destructive inflammation. Whether it be metoprolol’s reduction of microvascular
obstruction/reperfusion injury, anti-PSGL-1 reduction of ALI, or rhVim’s interaction
with P-selectin to reduce inflammation in an ALI model, all build off the same
concepts of neutrophil-platelet communication disruption to attenuate

Post Author: admin


I'm Jeremy!

Would you like to get a custom essay? How about receiving a customized one?

Check it out